Find resources for the original iBlot Gel Transfer Device, including details and performance data for the iBlot Western Detection Kits as well as application notes, FAQs, literature citations, ordering information, and more.

A: No. Do not use iBlot Transfer Stacks in the iBlot 2 Gel Transfer Device, and do not mix components between iBlot Transfer Stacks and iBlot 2 Transfer Stacks.

Q: Is there a limitation on the thickness of gels that can be used with the iBlot Dry Blotting System?

A: The iBlot Dry Blotting System has been tested to efficiently transfer protein from gels ranging in thickness from 1 mm to 3 mm. We have not tested gels thicker than 3 mm because they are rarely used for SDS-PAGE.

Q: I want to conduct western transfer with mini gels (8 x 8 cm), but I don’t have iBlotTransfer Stacks (Mini). Can I use iBlot Transfer Stacks (Regular) to transfer my mini gels?

A: It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible.

Q: How can we be more environmentally responsible?

A: The plastic in the iBlot Dry Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.

Q: Are the copper electrodes in the transfer stacks recyclable?

A: No. The electrodes are copper-coated nylon, and the amount of copper left in the electrode after a transfer is rather small. There is 0.6 grams of copper per sheet before transfer, and even less after transfer.

Q: Sometimes there is green discoloration on the blot around the gel. What is this, and does it affect the results?

A: The green discoloration is copper deposits from the iBlot Transfer Stacks, and it does not affect the results. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack. The current formulation of stacks minimizes the green discoloration.

A: No. The PVDF membrane comes preactivated. You just need to open the transfer stack with membrane, place the separation gel on top of the membrane, and apply one layer of moistened filter paper to run (the same as with the nitrocellulose stacks).

Q: Can iBlot Transfer Stacks be used more than once?

A: No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.

Q: What is the shelf life of both the nitrocellulose and PVDF iBlot Transfer Stacks?

A: The minimum guaranteed shelf life for iBlot Transfer Stacks is 2 months. Depending on when you purchase the transfer stack, shelf life will be 2–8 months.

Q: Is it possible to substitute the membrane from the iBlot Transfer Stack with my specialized membrane?

A: In theory, you can replace the membrane provided in your iBlot Transfer Stack with any membrane that is compatible with western blotting. To do this, cut the alternative membrane to match the size of your gel, and wet the membrane. Then either place the alternative membrane on top of the integrated membrane, or carefully remove the integrated membrane from the gel matrix with forceps and replace it with the new membrane. Note that Life Technologies only supports the use of iBlot Transfer Stacks when they are used with the provided instructions.

Q: Occasionally my western blots have high background. What do you recommend?

A: This may be a result of insufficient blocking or nonspecific binding. We suggest trying our
WesternBreeze Blocker/Diluent. We have been using it with good success. Additionally, you should optimize primary and secondary antibody concentrations as generally recommended for any new blotting technique. Many cases of high background can be resolved by further diluting one or both antibody preparations.

Q: How can I get better transfer of high molecular weight proteins?

A: Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from gel to membrane. We recommend extending the transfer time by 8–10 minutes for optimal transfer of proteins >150 kDa using the iBlot Dry Blotting System. To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE Novex 3-8% Tris-acetate gels for electrophoresis. We have an application note available, titled “Transferring Large and Small Proteins Using the iBlot 7-Minute Blotting System”
. To download the pdf, see Application notes tab on this page.

Q: What causes empty spots on my membrane after transfer?

A: The iBlot Dry Blotting System is similar to conventional transfer methods in that air bubbles between the gel and the membrane will prevent protein transfer. Be sure to remove all air bubbles between the gel and membrane before starting the transfer, using the blotting roller supplied with the iBlot Dry Blotting System.

Q: Does the iBlot Dry Blotting System work with native or native-blue gels?

A: Yes, we have an application note available, titled, “Western Blotting NativePAGE Novex Bis-Tris gels Using the iBlot 7-Minute Blotting System’. To download the pdf, see Application notes tab on this page.

Q: Do I need to have an iBlot Dry Blotting System to use an iBlot Western Detection Kit?

A: Yes. The iBlot Western Detection Kits use the iBlot system to produce an electrical field that accelerates interactions between antibodies and blocking reagents with membrane-bound antigens. iBlot Western Detection Kits require an iBlot Dry Blotting System with the P9 program (available in software versions 2.9.5 and higher). If you have an older iBlot Dry Blotting System, the firmware is freely available for download. See the
Instrument Registration page to find out how.

Q: Can iBlot Western Detection Stacks be used more than once?

A: No. The detection stacks have a finite amount of ions and are depleted after a single use, which includes the three detection steps: blocking, primary antibody, and secondary antibody.

Q: Should I change the detection stacks for each step?

A: No. A single set of stacks is used for all three steps of the western detection.

Q: Does it matter what method I use to transfer protein to my western blot membrane?

A: No, it does not matter. iBlot Western Detection Kits are compatible with western blots created using wet or semi-dry methods. You can use one regular-sized membrane (13.5 cm x 8 cm) or 1–2 mini-sized membranes (8 x 8 cm).

Q: How many reactions can I perform at one time?

A: iBlot Western Detection Stacks come in two sizes: mini and regular; they are supplied with assay spacers to create partitions for analysis using more than one set of antibodies. With the mini size you can perform detection on one mini-sized membrane or two halves of a mini-sized membrane. With the regular size you can perform detection on two mini-sized membranes or 4 quarters of a regular-sized membrane (4 x 8 cm).

Q: Sometimes I see high background on the membrane after detection. How can I eliminate it?

A: High background can be caused by nonspecific antibody binding. To eliminate it, use a lower concentration of the secondary antibody (as specified in the manual). In addition, using nitrocellulose rather than PVDF membranes will help to minimize background.

Q: Sometimes my molecular weight (MW) markers are no longer visible after my detection is completed. Does this mean my proteins are also gone?

A: Definitely not. MW markers are usually prestained, and the stains are more highly charged than typical cellular proteins. Applying an electrical field in the western detection procedure can drive the MW markers right through the membrane onto the bottom stack. Cellular proteins, however, are less charged than the MW markers, and at the detection stage they no longer contain any SDS. The result is that the cellular proteins stay immobilized on the membrane during western detection using the iBlot system.

Q: Can I use different antibodies or different dilutions of the same antibody in the procedure?

A: Yes. iBlot Western Detection Kits are supplied with reusable spacers, expressly for this purpose. Use spacers to separate membrane sections for detection with different antibody solutions at the same time. A detailed protocol is provided in the user manual.

Q: What membrane type should I use with the iBlot Western Detection Kits? Is one type preferred over another?

A: You can use nitrocellulose or PVDF membranes with the iBlot Western Detection Kits. However, we highly recommend using nitrocellulose rather than PVDF because less background is seen with nitrocellulose membranes. This results in more sensitive detection.

Q: Can I use the iBlot Western Detection Kits on membranes with previously transferred proteins?

A: Yes, as long as you activate the membrane before you start the detection procedure. Completely wet nitrocellulose membranes using water. Activate PVDF membranes by immersing in 100% methanol for several seconds until the membrane is completely wet, then rinse with water. Then proceed with the iBlot Western Detection Kit.

Q: What substrate is used with the kits?

A: The substrate used for the iBlot Western Detection Kits reacts with alkaline phosphatase (AP). Therefore, horseradish peroxidase (HRP) substrates will not work for this kit. Other conjugates and substrates of AP cannot be used with iBlot Western Detection Kits. The reagents supplied with the kits have been optimized to obtain the best results.

Q: How is it possible to finish the entire immunodetection in just 30 minutes?

A: Native antibodies typically have some negative charge at neutral pH, which allows their electrophoretic migration in low voltage from the carrier matrix onto the membrane. The force of the electric field is counterbalanced by their affinity for antigen, so that binding antibodies are “captured” while unbound antibodies flow through the blocked membrane and onto the bottom stack. Basically, the iBlot Dry Blotting System electrophoretically focuses antibodies to greatly accelerate their interaction rate with immobilized antigens.

Q: Are the kits compatible with the Li-COR® system?

A: Currently, the iBlot Western Detection Kits use a green matrix, and unfortunately this can create background fluorescence that isn’t compatible with the Li-COR Odyssey scanner.

Q: How can I improve the signal intensity?

A: The signal intensity is primarily controlled by the concentration of the secondary antibody; nonspecific binding can result when the concentration is too high, and poor binding to the primary antibody can result when the solution is too dilute. The next option is to modify the primary antibody concentration, which controls the number of antibody molecules that bind to the antigen, and thus the signal and background levels. (See the manual for further details.)

Q: Can the primary antibody solution be reused?

A: No. The primary antibody solution cannot be reused after it has been applied to the matrix.

Q: What concentration of primary antibody should I use with the iBlot Western Detection Kits?

A: Use twice the primary antibody concentration used in typical western blot detection procedures. Note that the overall amount of the primary antibody used is the same as in typical traditional methods, because only half the volume of primary antibody solution is needed with the iBlot Western Detection System.

Q: What is the shelf life of the iBlot Western Detection Stacks?

A: The shelf life is 6 months, at room temperature.

Q: What is the shelf life of the iBlot Western Detection Reagents?

A: The shelf life is 12 months, at 4°C.

Q: I have reagents left but the detection stacks have expired. Can I purchase only the iBlot Western Detection Stacks?

Dobbertin A et al. 2009. Targeting of acetylcholinesterase in neurons in vivo: a dual processing function for the proline-rich membrane anchor subunit and the attachment domain on the catalytic subunit.
J Neurosci 29:4519–4530.

Elmen J et al. 2008. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver.
Nucleic Acids Res 36:1153–1162.

Fast transfer

Useful and convenient

I remember the days when it took hours to do a western blot. Now with the iBlot, it takes only 7 minutes! You save a ton of time sitting around; instead, you can get on with your experiments/results in less than 10 minutes. It's quick and gives you just as good a blot as any old western blot technique. But I do agree with another reviewer that the device can get a bit hot, therefore "frying" the gel. The gel may get really warm but so far that hasn't affected any of my experiments.

Date: February 5, 2010Reviewer: NameInstitution: Kalobios

Increases blot throughput

The iBlot has been a great addition to the lab. It is very easy to use, transfers very quickly, and is mess-free with its no buffer system. Overall, I have been very happy with it. The only issues I have are when I'm transferring very small proteins (~15 kDa) I have incomplete transfers, for these I still use the Invitrogen blot module. Otherwise, it is a great set up.

Date: February 5, 2010Reviewer: CarlyInstitution: Repligen

Fast and convenient

Easy and fast transfers with no need for transfer buffers. Not quite as good with very large molecular weight proteins.

A: No. Do not use iBlot Transfer Stacks in the iBlot 2 Gel Transfer Device, and do not mix components between iBlot Transfer Stacks and iBlot 2 Transfer Stacks.

Q: Is there a limitation on the thickness of gels that can be used with the iBlot Dry Blotting System?

A: The iBlot Dry Blotting System has been tested to efficiently transfer protein from gels ranging in thickness from 1 mm to 3 mm. We have not tested gels thicker than 3 mm because they are rarely used for SDS-PAGE.

Q: I want to conduct western transfer with mini gels (8 x 8 cm), but I don’t have iBlotTransfer Stacks (Mini). Can I use iBlot Transfer Stacks (Regular) to transfer my mini gels?

A: It is best not to transfer a single mini gel on a regular-sized transfer stack. Although in most cases the transfer will be fine, empty spaces on the transfer stack that are not in direct contact with a gel could potentially cause distortions across the whole surface of the membrane, including the portion in contact with the gel. It is best to have more than 50% of a membrane in contact with the gel, if possible.

Q: How can we be more environmentally responsible?

A: The plastic in the iBlot Dry Blotting System stack packaging is polyethylene terephthalate (PET) and can be recycled.

Q: Are the copper electrodes in the transfer stacks recyclable?

A: No. The electrodes are copper-coated nylon, and the amount of copper left in the electrode after a transfer is rather small. There is 0.6 grams of copper per sheet before transfer, and even less after transfer.

Q: Sometimes there is green discoloration on the blot around the gel. What is this, and does it affect the results?

A: The green discoloration is copper deposits from the iBlot Transfer Stacks, and it does not affect the results. To minimize this effect, shake excess water off the filter paper and buffer from the gel before placing each on the stack. The current formulation of stacks minimizes the green discoloration.

A: No. The PVDF membrane comes preactivated. You just need to open the transfer stack with membrane, place the separation gel on top of the membrane, and apply one layer of moistened filter paper to run (the same as with the nitrocellulose stacks).

Q: Can iBlot Transfer Stacks be used more than once?

A: No. The transfer stacks have a finite amount of ions to drive the transfer and are depleted after a single use.

Q: What is the shelf life of both the nitrocellulose and PVDF iBlot Transfer Stacks?

A: The minimum guaranteed shelf life for iBlot Transfer Stacks is 2 months. Depending on when you purchase the transfer stack, shelf life will be 2–8 months.

Q: Is it possible to substitute the membrane from the iBlot Transfer Stack with my specialized membrane?

A: In theory, you can replace the membrane provided in your iBlot Transfer Stack with any membrane that is compatible with western blotting. To do this, cut the alternative membrane to match the size of your gel, and wet the membrane. Then either place the alternative membrane on top of the integrated membrane, or carefully remove the integrated membrane from the gel matrix with forceps and replace it with the new membrane. Note that Life Technologies only supports the use of iBlot Transfer Stacks when they are used with the provided instructions.

Q: Occasionally my western blots have high background. What do you recommend?

A: This may be a result of insufficient blocking or nonspecific binding. We suggest trying our
WesternBreeze Blocker/Diluent. We have been using it with good success. Additionally, you should optimize primary and secondary antibody concentrations as generally recommended for any new blotting technique. Many cases of high background can be resolved by further diluting one or both antibody preparations.

Q: How can I get better transfer of high molecular weight proteins?

A: Proteins larger than ~150 kDa migrate more slowly than smaller proteins. Therefore, more time is required to transfer them from gel to membrane. We recommend extending the transfer time by 8–10 minutes for optimal transfer of proteins >150 kDa using the iBlot Dry Blotting System. To enhance transfer efficiency, we also recommend adding an equilibration (gel-soaking) step between electrophoresis and western transfer and using NuPAGE Novex 3-8% Tris-acetate gels for electrophoresis. We have an application note available, titled “Transferring Large and Small Proteins Using the iBlot 7-Minute Blotting System”
. To download the pdf, see Application notes tab on this page.

Q: What causes empty spots on my membrane after transfer?

A: The iBlot Dry Blotting System is similar to conventional transfer methods in that air bubbles between the gel and the membrane will prevent protein transfer. Be sure to remove all air bubbles between the gel and membrane before starting the transfer, using the blotting roller supplied with the iBlot Dry Blotting System.

Q: Do I need to have an iBlot Dry Blotting System to use an iBlot Western Detection Kit?

A: Yes. The iBlot Western Detection Kits use the iBlot system to produce an electrical field that accelerates interactions between antibodies and blocking reagents with membrane-bound antigens. iBlot Western Detection Kits require an iBlot Dry Blotting System with the P9 program (available in software versions 2.9.5 and higher). If you have an older iBlot Dry Blotting System, the firmware is freely available for download. See the
Instrument Registration page to find out how.

Q: Can iBlot Western Detection Stacks be used more than once?

A: No. The detection stacks have a finite amount of ions and are depleted after a single use, which includes the three detection steps: blocking, primary antibody, and secondary antibody.

Q: Should I change the detection stacks for each step?

A: No. A single set of stacks is used for all three steps of the western detection.

Q: Does it matter what method I use to transfer protein to my western blot membrane?

A: No, it does not matter. iBlot Western Detection Kits are compatible with western blots created using wet or semi-dry methods. You can use one regular-sized membrane (13.5 cm x 8 cm) or 1–2 mini-sized membranes (8 x 8 cm).

Q: How many reactions can I perform at one time?

A: iBlot Western Detection Stacks come in two sizes: mini and regular; they are supplied with assay spacers to create partitions for analysis using more than one set of antibodies. With the mini size you can perform detection on one mini-sized membrane or two halves of a mini-sized membrane. With the regular size you can perform detection on two mini-sized membranes or 4 quarters of a regular-sized membrane (4 x 8 cm).

Q: Sometimes I see high background on the membrane after detection. How can I eliminate it?

A: High background can be caused by nonspecific antibody binding. To eliminate it, use a lower concentration of the secondary antibody (as specified in the manual). In addition, using nitrocellulose rather than PVDF membranes will help to minimize background.

Q: Sometimes my molecular weight (MW) markers are no longer visible after my detection is completed. Does this mean my proteins are also gone?

A: Definitely not. MW markers are usually prestained, and the stains are more highly charged than typical cellular proteins. Applying an electrical field in the western detection procedure can drive the MW markers right through the membrane onto the bottom stack. Cellular proteins, however, are less charged than the MW markers, and at the detection stage they no longer contain any SDS. The result is that the cellular proteins stay immobilized on the membrane during western detection using the iBlot system.

Q: Can I use different antibodies or different dilutions of the same antibody in the procedure?

A: Yes. iBlot Western Detection Kits are supplied with reusable spacers, expressly for this purpose. Use spacers to separate membrane sections for detection with different antibody solutions at the same time. A detailed protocol is provided in the user manual.

Q: What membrane type should I use with the iBlot Western Detection Kits? Is one type preferred over another?

A: You can use nitrocellulose or PVDF membranes with the iBlot Western Detection Kits. However, we highly recommend using nitrocellulose rather than PVDF because less background is seen with nitrocellulose membranes. This results in more sensitive detection.

Q: Can I use the iBlot Western Detection Kits on membranes with previously transferred proteins?

A: Yes, as long as you activate the membrane before you start the detection procedure. Completely wet nitrocellulose membranes using water. Activate PVDF membranes by immersing in 100% methanol for several seconds until the membrane is completely wet, then rinse with water. Then proceed with the iBlot Western Detection Kit.

Q: What substrate is used with the kits?

A: The substrate used for the iBlot Western Detection Kits reacts with alkaline phosphatase (AP). Therefore, horseradish peroxidase (HRP) substrates will not work for this kit. Other conjugates and substrates of AP cannot be used with iBlot Western Detection Kits. The reagents supplied with the kits have been optimized to obtain the best results.

Q: How is it possible to finish the entire immunodetection in just 30 minutes?

A: Native antibodies typically have some negative charge at neutral pH, which allows their electrophoretic migration in low voltage from the carrier matrix onto the membrane. The force of the electric field is counterbalanced by their affinity for antigen, so that binding antibodies are “captured” while unbound antibodies flow through the blocked membrane and onto the bottom stack. Basically, the iBlot Dry Blotting System electrophoretically focuses antibodies to greatly accelerate their interaction rate with immobilized antigens.

Q: Are the kits compatible with the Li-COR® system?

A: Currently, the iBlot Western Detection Kits use a green matrix, and unfortunately this can create background fluorescence that isn’t compatible with the Li-COR Odyssey scanner.

Q: How can I improve the signal intensity?

A: The signal intensity is primarily controlled by the concentration of the secondary antibody; nonspecific binding can result when the concentration is too high, and poor binding to the primary antibody can result when the solution is too dilute. The next option is to modify the primary antibody concentration, which controls the number of antibody molecules that bind to the antigen, and thus the signal and background levels. (See the manual for further details.)

Q: Can the primary antibody solution be reused?

A: No. The primary antibody solution cannot be reused after it has been applied to the matrix.

Q: What concentration of primary antibody should I use with the iBlot Western Detection Kits?

A: Use twice the primary antibody concentration used in typical western blot detection procedures. Note that the overall amount of the primary antibody used is the same as in typical traditional methods, because only half the volume of primary antibody solution is needed with the iBlot Western Detection System.

Q: What is the shelf life of the iBlot Western Detection Stacks?

A: The shelf life is 6 months, at room temperature.

Q: What is the shelf life of the iBlot Western Detection Reagents?

A: The shelf life is 12 months, at 4°C.

Q: I have reagents left but the detection stacks have expired. Can I purchase only the iBlot Western Detection Stacks?

Dobbertin A et al. 2009. Targeting of acetylcholinesterase in neurons in vivo: a dual processing function for the proline-rich membrane anchor subunit and the attachment domain on the catalytic subunit.
J Neurosci 29:4519–4530.

Elmen J et al. 2008. Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver.
Nucleic Acids Res 36:1153–1162.

Fast transfer

Useful and convenient

I remember the days when it took hours to do a western blot. Now with the iBlot, it takes only 7 minutes! You save a ton of time sitting around; instead, you can get on with your experiments/results in less than 10 minutes. It's quick and gives you just as good a blot as any old western blot technique. But I do agree with another reviewer that the device can get a bit hot, therefore "frying" the gel. The gel may get really warm but so far that hasn't affected any of my experiments.

Date: February 5, 2010Reviewer: NameInstitution: Kalobios

Increases blot throughput

The iBlot has been a great addition to the lab. It is very easy to use, transfers very quickly, and is mess-free with its no buffer system. Overall, I have been very happy with it. The only issues I have are when I'm transferring very small proteins (~15 kDa) I have incomplete transfers, for these I still use the Invitrogen blot module. Otherwise, it is a great set up.

Date: February 5, 2010Reviewer: CarlyInstitution: Repligen

Fast and convenient

Easy and fast transfers with no need for transfer buffers. Not quite as good with very large molecular weight proteins.

Western detection kits for use with the iBlot Gel Transfer Device

Easy optimization—allows the use of different conditions for different sections of the blot

Optimized components—kit contains the reagents and detection stacks needed for western detection; all you supply is the primary antibody

Designed to be compatible with the original iBlot Gel Transfer Device, iBlot Western Detection Kits offer complete western detection in less than 25 minutes with sensitivity that is equal to (or better than) conventional protocols for most antibody–antigen pairs. The kits are available with anti-mouse or anti-rabbit secondary antibodies and are compatible with chemiluminescent and chromogenic detection.